The cornea is an important tissue not only composing the wall of the eyeball with the sclera, but also behaving as an entrance to take in an image of the outside into the eye by virtue of a transparent tissue. Unlike other biotissues, the cornea has no blood vessel and is transparent, and both of the surfaces are smooth and spherical. The outer surface of the cornea is covered with the tear film, and the inner surface thereof contacts with the anterior chamber that is filled with aqueous humor.
The cornea has a thickness of approximately 500 μm at the central part, and consists of five layers of, starting from the outside, corneal epithelium, Bowman membrane, stroma, Descemet membrane, and corneal endothelium. The corneal epithelial layer has a thickness of approximately 10% of the whole thickness of the cornea, and is formed of corneal epithelial cells. When the corneal epithelium is injured, peripheral epithelial cells migrate to the injured part and proliferate there to repair damage. The thickness of the corneal stroma accounts for approximately 90% of the whole thickness of the cornea. The main component of the corneal stroma is collagen, and besides, the corneal stroma contains mucopolysaccharide. The mucopolysaccharide is water absorptive, and tends to bulging (swelling) by absorbing water. However, the water content in the corneal stroma is maintained constant due to the pumping function and barrier function of the corneal endothelium.
The corneal endothelium is composed of corneal endothelial cells. The corneal endothelium cannot regenerate once it falls away because human corneal endothelial cells do not proliferate. When human corneal endothelial cells are injured and then fall away, the defect cannot be repaired by the cell proliferation. Alternatively, peripheral endothelial cells will migrate to and enlarge in the defect area and then form new cell adhesion, thereby compensating the defect. Therefore, the adhesive property of a human corneal endothelial cell is an important function of repairing the injured part. In addition, since the number of corneal endothelial cells is limited, when the corneal endothelium is suffered serious injury the corneal endothelium cells cannot repair it and thus the defect remains in the corneal endothelium. The defect formed in the corneal endothelium leads to irreversible serious dysfunction. Specifically, when the density of corneal endothelial cells is approximately 500 cells/mm2 or less, it is impossible to repair the defect in the corneal endothelium, causing corneal edema (bullous keratopathy) associated with irreversible opacity. Note that it is considered that the density of normal corneal endothelial cells is approximately 2500 to 3000 cells/mm2.
A corneal endothelial cell has the pumping function of draining water from the corneal stroma which tends to swelling by absorbing the water into the anterior chamber, and the barrier function of controlling transfer of the water from the anterior chamber to the corneal stroma. When these functions of corneal endothelial cells decrease, the corneal stroma will contain excessive water, thereby not only increasing the thickness of the cornea, but also causing edema (bullous keratopathy) associated with irreversible opacity, with the result that the visual acuity will extremely decrease. Because swelling of the cornea increases the thickness of the cornea and leads to occurrence of edema, it is necessary to thin the cornea and to prevent the cornea from swelling.
The dysfunction of the corneal endothelium is considered to account for approximately 60% of the whole dysfunction of the cornea. The defect in the corneal endothelium due to physical injury, progressive corneal endothelial dystrophy (typically Fuchs corneal dystrophy) or the like results in bullous keratopathy associated with irreversible corneal edema. When the visual acuity extremely decreases, corneal transplant is required.
Although human corneal endothelial cells do not proliferate in vivo, it has been examined to culture them in vitro (see Non-Patent Documents 1 and 2). This is because it is possible to treat the cornea by transplanting human corneal endothelial cells which have been cultured and proliferated in vitro.
Thus, development is desired of not only a medical agent for enhancing the adhesive property of a corneal endothelial cell, and furthermore for repairing the injured part of the corneal endothelium or for maintaining and recovering the functions of the corneal endothelium, but also a culture medium for culturing and proliferating a human corneal endothelial cell, a preservation solution for storing a corneal endothelial cell or a corneal endothelial tissue until transplant, a medical agent for modulating the thickness of the cornea, and the like.
Rho kinase (Rho-associated, coiled-coil containing protein kinase: ROCK) is a serine threonine kinase having a molecular weight of approximately 160 kDa, and its gene is highly conserved from lower animals such as nematode and drosophila to humans. Rho kinase contributes to physiological functions such as contraction of smooth-muscle cells, control of cell morphology, migration, and control of gene expression. Development is promoted of Rho kinase inhibitor as a therapeutic agent for circulatory disease and the like.
In addition, in recent years, development thereof has been made as a medicine for topical administration for use in treatment of eye diseases such as glaucoma. Furthermore, as to culture of a corneal endothelial cell, it has been reported that a rabbit corneal endothelial cell can be cultured in the presence of (+)-trans-4-(1-aminoethyl)-1-(4-pyridylaminocarbonyl)cyclohexane (hereinafter referred to as Y-27632) that is one kind of Rho kinase inhibitor (see Non-Patent Documents 3 and 4), and it has been reported that a monkey corneal endothelial cell can also be cultured in the same way (see Patent Document 1). Patent Document 1 reports that the adhesive property of a rabbit corneal endothelial cell is enhanced also with 1-(5-isoquinolinesulfonyl)-1,4-homopiperazine) hereinafter referred to as fasudil). Moreover, treatment of injured rabbit corneal endothelium with Y-27632 (see Non-Patent Document 5) and treatment of human Fuchs corneal dystrophy with Y-27632 (see Non-Patent. Document 6) have been reported. In addition, treatment of injured rabbit corneal endothelium with (R)—(+)—N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4-(1-aminoethyl)benzamide (hereinafter, referred to as Y-39383) also has been reported (see Patent Document 2 and Non-Patent Document 7). According to Non-Patent Document 7, the 50% inhibitory concentrations (IC50) of Y-33983 and Y-27632 for Rho kinase are 0.0036 μM and 0.11 μM, respectively, between which there is an approximately 30 times difference. The difference is considered to result in a difference of activity for a corneal endothelial cell.
Both Y-27632 and Y-39983 used in these Documents are N-pyridyl amide compounds, whereas fasudil is a 1-sulfone-1,4-homopiperazine compound.
On the other hand, it has been disclosed that 1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine (hereinafter, also referred to as Compound A) is a 1-sulfone-1,4-homopiperazine compound like fasudil, but has a higher selectivity than that of fasudil, and has a higher Rho kinase inhibitory activity than that of fasudil, and it has been reported that the compound is useful for prevention or treatment of asthma (see Compound 6 in Patent Document 3). Patent Document 3 discloses that the 50% inhibitory concentrations (IC50)of Compound A and fasudil for Rho kinase are 0.2 μM and 1.5 μM, respectively. In comparison with the disclosure in above-mentioned Non-Patent Document 7, the 50% inhibitory concentration (IC50) of Compound A for Rho kinase is considered to be more potent than that of fasudil, but less potent than or similar to that of Y-27632.
As to use of Compound A for eye diseases, it has been reported that combination of Compound A with carbonic anhydrase inhibitor is useful for prevention or treatment of glaucoma (see Patent Document 4), and Compound A is useful for prevention or treatment of ocular fundus disease (see Patent Document 5), but the effect of Compound A on the corneal thickness has not been reported.